Heterologous expression of the biosynthetic gene cluster of pamamycin and investigation on the functions of regulatory genes from Streptomyces lincolnensis NRRL 2936

Abstract:

[Background] Pamamycins are macrolid compounds with anti-infectin bioactivities. Their unique structural features and promising biological activity stimulated extensive efforts towards their various investigations. Furtehermore, we discovered that the biosynthetic gene cluster of pamamycins was located in the chromosome of Streptomyces lincolnensis NRRL 2936. [Objective] The biosynthetic pathway had been investigated, and the function of two regulatory genes in the gene cluster was elusive. In this research, the plasmid pJQK450 containing a full length of pamamycin biosynthetic gene cluster was cloned from the genomic library of S. lincolnensis NRRL 2936 and introduced into heterologous hosts by intergeneric conjugation. Subsequently, the function of two regulatory genes was elucidated by targeted gene disruption and complementaion. [Methods] The Fosmid, named pJQK450, containing of the completed pamamycin gene cluster was captured by narrow-down polymerase chain reaction strategy from Streptomyces lincolnensis NRRL 2936 Fosmid genome library. Plasmid pJQK450 was transferred into the E. coli ET12567/pUZ8002 and then introduced into the heterologous hosts by intergeneric conjugation. Then, the validated-exconjugants were fermented, and the biosynthetic capacity of pamamycin of the recombinant strains was evaluated by bioactivity analysis against Mycobacteriun smegmatis mc2155 and LC-MS detection. Furthermore, the regulation roles of PamR1 and PamR2 were determined by targeted gene inactivation and complementation experiments. [Results] The entire gene cluster of pamamycin was successful expressed in S. coelicolor M1154. Through genetic validation, both PamR1 and PamR2 were found to be negative regulators in pamamycin biosynthesis. [Conclusion] Heterologous expression of pamamycin facilitates the biosynthesis engineering and targeted accumulating pamamycin component. Additionally, the determination of the two negative regulatory genes paved way for the yield improvement of pamamycin.